The long-term objectives of this proposal are to determine the magnitude of PGRMC1's effect on CYP3A4 activity and if this effect is substrate specific. PGRMC1 is a hemoprotein with a cytochrome b5-like domain. PGRMC1 enhances CYP activity in yeast and in mammalian adrenal cells but it is unknown how or if it interacts and affects activity of drug metabolizing cytochromes P450. The central hypothesis to be tested is that PGRMC1 modulates CYP3A4 catalysis. We propose this novel idea because both PGRMC1 and CYP3A4 co-localize in the hepatic endoplasmic reticulum, and because PGRMC1 physically associates with CYP3A4. Nonetheless, PGRMC1's role in modulating CYP3A4 catalytic activity remains unexplored. Our preliminary data further reveal a relationship between PGRMC1 and CYP3A4 activity by showing, among 530 human livers, a significant correlation between CYP3A4 mediated metabolism of both midazolam and testosterone and PGRMC1 mRNA expression, and a PGRMC1 associated genotype. We propose to test the hypothesis that PGRMC1 modulates CYP3A4 activity in three specific aims. In Aim 1, the extent of PGRMC1's effect on CYP3A4 activity will be quantified. CYP3A4 mediated metabolism of testosterone and midazolam will be compared between (a) CYP3A4/POR (human NADPH-P450 reductase) supersomes with and without expressed PGRMC;(b) CYP3A4/POR supersomes reconstituted with purified PGRMC1;and (c) pooled human liver microsomes in which PGRMC1 activity is decreased with PGRMC1 inhibitory antibodies. To determine if the effect is tissue specific, in Aim 2 testosterone and midazolam oxidation will be compared among Adenovirus-CYP3A4/POR transfected human liver, intestine and kidney cells in which PGRMC1 expression is increased with AdV-PGRMC1 or depleted with PGRMC1 ShRNA lentivirus. In Aim 3 the requirement for PGRMC1's heme binding domain will be determined by mutating it and examining the effects on PGRMC1's ability to interact with CYP3A4 and modulate its activity. In total the results should provide novel and important information about the contribution of PGRMC1 to CYP3A catalytic activity in liver, intestine and kidney cells. PUBLIC HEALTH RELEVANCE: CYP3A4 oxidatively metabolizes over half of the orally effective drugs in use today. Despite the fact that PGRMC1 is required for yeast CYP activity, modulates mammalian adrenal CYP activity, and has been shown to directly interact with CYP3A4, its contribution to CYP3A4 activity has never been tested. A clearer understanding of the role PGRMC1 plays in modulating CYP3A4 activity should enhance the development of safer medications. Specifically, gaining an understanding of the role of PGRMC1 in modulating CYP3A4 catalytic activity will increase our ability to accurately predict the metabolic rate and metabolite profiles of drugs in patients using improved in vitro systems. Moreover, it is likely that modulation of CYP3A4 activity by PGRMC1 will be a paradigm for its interactions with other xenobiotic metabolizing CYPs.